Amplified and overexpressed gene in colorectal cancers

ABSTRACT

This invention relates to methods to diagnose colon cancer and other proliferative diseases. Gene 26#77 is identified herein as a novel oncogene. Methods are provided for diagnosing and treating a disease or disorder characterized by amplification of the 26#77 gene and/or overexpression of 26#77 gene products. The 26#77 gene is located on chromosome 20q13.2, a region whose amplification is associated with a poor cancer prognosis. The 26#77 gene is amplified and 26#77 RNA and protein are overexpressed in 60% of colorectal cancers.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support by Grant No. CA32737,awarded by the National Institutes of Health. The Government has certainrights in this invention.

FIELD OF THE INVENTION

This invention relates to methods to diagnose colon cancer and otherproliferative diseases.

BACKGROUND OF THE INVENTION

Chromosome abnormalities are often associated with genetic disorders,degenerative diseases, and cancer. The deletion or multiplication ofcopies of whole chromosomes and the deletion or amplifications ofchromosomal segments or specific regions are common occurrences incancer (Smith (1991) Breast Cancer Res. Treat. 18: Suppl. 1:5-14; van deVijer (1991) Biochim. Biophys. Acta. 1072:33-50). In fact,amplifications and deletions of DNA sequences can be the cause of acancer. For example, proto-oncogenes and tumor-suppressor genes,respectively, are frequently characteristic of tumorigenesis (Dutrillaux(1990) Cancer Genet. Cytogenet. 49: 203-217). Clearly, theidentification and cloning of specific genomic regions associated withcancer is crucial both to the study of tumorigenesis and in developingbetter means of diagnosis and prognosis.

One of the amplified regions found in studies of breast and colon cancercells is on chromosome 20, specifically, 20q13.2 (see, e.g. WO98/02539).Amplification of 20q13.2 was subsequently found to occur in a variety oftumor types and to be associated with aggressive tumor behavior.Increased 20q13.2 copy number has been found in 40% of breast cancercell lines and 18% of primary breast tumors (Kalliioniemi (1994) Proc.Natl. Acad. Sci. USA 91: 2156-2160). Copy number gains at 20q13.2 havealso been reported in greater than 25% of cancers of the ovary (Iwabuchi(1995) Cancer Res. 55:6172-6180), colon (Schlegel (1995) Cancer Res. 55:6002-6005 and WO02/06526), head-and-neck (Bockmuhl (1996) Laryngor. 75:408-414), brain (Mohapatra (1995) Genes Chromosomes Cancer 13: 86-93),pancreas (Solinas-Toldo (1996) Genes Chromosomes Cancer 20:399-407).

A number of studies have elucidated genetic alterations that occurduring the development of colorectal tumors. For instance, deletions ofp53 genes on chromosome 17p are often late events associated with thetransition from the benign (adenoma) to the malignant (carcinoma) state.See Vogelstein et al., New England Journal of Medicine, 319:525 (1988),Fearon and Vogelstein, Cell, 61:759-767 (1990) and Baker et al. CancerRes. 50:7717-22 (1990). More recently, comparative genomic hybridizationhas shown that specific patterns of chromosomal gains and losses takeplace during colorectal carcinogenesis (see, e.g. Schlegel, et al.Cancer Research. 55, 6002-6005 (1995); Ried, et al. Genes, Chromosomes &Cancer 15, 234-245 (1996); and Nakao et al., Jpn. J. Surg. 28, 567-569(1998). These changes included overrepresentation (amplification) oflarge portion of chromosome 20 material.

The identification of new genes that are responsible for carcinogenesisis obviously great use in diagnosis, prognosis and treatment of thesediseases. The present invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

This invention provides a method for determining the presence or absenceof a colorectal cancer cell in a patient, by determining the level of atarget nucleic acid that encodes the 26#77 protein (e.g., SEQ ID NO: 2)in a biological sample from the patient. In one embodiment, the targetnucleic acid comprises a sequence at least 80% identical to SEQ IDNO: 1. In a further embodiment, the biological sample can includeisolated nucleic acids. In another embodiment, the nucleic acids areamplified before the level of the target nucleic acid is determined. Inan additional embodiment the isolated nucleic acids are mRNA.

In one aspect, the biological sample is colorectal tissue and the stepof determining the level of target nucleic acid is carried out using insitu hybridization.

In another aspect, the step of determining the level of target nucleicacid is carried out using a labeled nucleic acid probe that selectivelyhybridizes to SEQ ID NO: 1 under stringent hybridization conditions. Thenucleic acid probe can be immobilized to a solid support. In a furtheraspect, the step of determining the level of target nucleic acid iscarried out using Northern blot analysis.

In one embodiment, the step of determining the level of the targetnucleic acid is carried out by comparing the amount of the targetnucleic acid in the biological sample to the amount of the targetnucleic acid in a reference sample. The reference sample can be fromnormal colorectal tissue.

In another embodiment, the levels of 26#77 encoding nucleic acid aredetermined when the patient is undergoing a therapeutic regimen to treatcolorectal cancer. The levels of 26#77 encoding nucleic acid can also bedetermined when the patient is suspected of having colorectal cancer.

In one embodiment this invention provides an isolated expression vectorwith a nucleic acid sequence that encodes SEQ ID NO: 2, the 26#77protein. In further embodiments, the nucleic acid sequence is at least80% identical to SEQ ID NO: 1. The invention also provides a host cellcontaining a vector that expresses a nucleic acid that encodes the 26#77protein.

In one embodiment this invention provides a method for determining thepresence or absence of a colorectal cancer cell in a patient, bydetermining the level of a target protein, the 26#77 protein includingthe sequence shown in SEQ ID NO: 2. Levels of the 26#77 protein aredetermined in a biological sample from the patient, thereby determiningthe presence or absence of the colorectal cancer cell in the patient. Inone aspect the 26#77 protein levels are determined by using an antibodyspecific for the 26#77 protein. The antibody can be a polyclonalantibody or a monoclonal antibody. In a further aspect, the antibody canbe labeled and the label can be a fluorescent label.

In a further embodiment, the step of determination of the level of the26#77 protein is carried out by comparing the amount of the 26#77protein in the biological sample to the amount of the 26#77 protein in areference sample. In one aspect, the reference sample is from normalcolorectal tissue. In another aspect, the determination of 26#77 proteinlevel is made when the patient is undergoing a therapeutic regimen totreat colorectal cancer. In a further aspect, the determination of 26#77protein level is made when the patient is suspected of having colorectalcancer.

In one embodiment, the present invention provides a method for treatinga cancer that overexpresses a 26#77 gene product by administering atherapeutically effective amount of an inhibitor of 26#77 gene productto a patient who a cancer that overexpresses 26#77. The inhibitor of the26#77 gene product can be an antisense RNA molecule, or an inhibitoryRNA molecule.

Definitions

The phrase “determining the level of a target nucleic acid” refers toany method that can be used to detect increased copy number of a genomicsequence or increased expression level of a target gene. Methods fordetermining increased copy number are well known and include nucleicacid hybridization methods as described below. Methods for determiningthe level of expression of a particular gene are well known in the art.Such methods include RT-PCR, real-time PCR, use of antibodies againstthe gene products, and the like. As explained below, methods of theinvention are used to detect increased copy number or overexpression ofa gene referred to here as 26#77. Typically, overexpression of aparticular gene is at least about 2 times, usually at least about 5times the level of expression in a normal cell from the same tissue.

The terms “26#77 protein” or “26#77polynucleotide” or refer to nucleicacid and polypeptide polymorphic variants, alleles, mutants, andinterspecies homologues of SEQ ID NO: 1 or SEQ ID NO: 2. Typically suchgenes or proteins have a sequence that has greater than about 70%nucleotide sequence identity, usually 80%, 85%, 90% or 99% or greatersequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, preferably over aregion of over a region of at least about 25, 50, 100, 200, 500, 1000,or more residues. A polynucleotide or polypeptide sequence is typicallyfrom a mammal including, but not limited to, primate, e.g., human;rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or othermammal. These terms include both naturally occurring or recombinantforms.

A “biological sample” as used herein is a sample of biological tissue orfluid that contains nucleic acids or polypeptides, e.g., of a 26#77protein, polynucleotide or transcript. Such samples include, but are notlimited to, tissue isolated from humans, or rodents, e.g., mice, andrats. Biological samples may also include sections of tissues such asbiopsy and autopsy samples, frozen sections taken for histologicpurposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin,etc. Biological samples also include explants and primary and/ortransformed cell cultures derived from patient tissues. A biologicalsample is typically obtained from a eukaryotic organism, most preferablya mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; arodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; orfish. Livestock and domestic animals are of particular interest.

“Providing a biological sample” means to obtain a biological sample foruse in methods described in this invention. Most often, this will bedone by removing a sample of cells from an animal, but can also beaccomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods of the invention in vivo. Archival tissues,having treatment or outcome history, will be particularly useful.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., greater than about 60% amino acid sequence identity, 65%, 70%,75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%, or higher identity over a specified region, when compared andaligned for maximum correspondence over a comparison window ordesignated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions, as well as naturally occurring, e.g.,polymorphic or allelic variants, and man-made variants. As describedbelow, the preferred algorithms can account for gaps and the like.Preferably, identity exists over a region that is at least about 25amino acids or nucleotides in length, or more preferably over a regionthat is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, bythe homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443-453, by the search for similarity method of Pearson andLipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444-2448, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Ausubel, et al. (eds. 1995 and supplements)Current Protocols in Molecular Biology Lippincott.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Nat'l Acad. Sci. USA 90:5873-5887). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. Log valuesmay be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110,150, 170, etc.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, e.g., where the two peptides differonly by conservative substitutions. Another indication that two nucleicacid sequences are substantially identical is that the two molecules ortheir complements hybridize to each other under stringent conditions, asdescribed below. Yet another indication that two nucleic acid sequencesare substantially identical is that the same primers can be used toamplify the sequences.

A “host cell” is a naturally occurring cell or a transformed cell thatcontains an expression vector and supports the replication or expressionof the expression vector. Host cells may be cultured cells, explants,cells in vivo, and the like. Host cells may be prokaryotic cells such asE. coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells, such as CHO, HeLa, and the like (see, e.g., theAmerican Type Culture Collection catalog or web site).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein or nucleic acid that is thepredominant species present in a preparation is substantially purified.In particular, an isolated nucleic acid is separated from some openreading frames that naturally flank the gene and encode proteins otherthan protein encoded by the gene. The term “purified” in someembodiments denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure. “Purify” or“purification” in other embodiments means removing at least onecontaminant from the composition to be purified. In this sense,purification does not require that the purified compound be homogenous,e.g., 100% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG, and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. In certain contexts each codon in anucleic acid (except AUG, which is ordinarily the only codon formethionine, and TGG, which is ordinarily the only codon for tryptophan)can be modified to yield a functionally identical molecule. Accordingly,a silent variation of a nucleic acid which encodes a polypeptide isimplicit in a described sequence with respect to the expression product,but not necessarily with respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions, or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds, or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. Typically conservativesubstitutions for one another: 1) Alanine (A), Glycine (G); 2) Asparticacid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton (1984) Proteins Freeman).

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. Oligonucleotides are typically less than about 100 nucleotidesin length. A nucleic acid of the present invention will generallycontain phosphodiester bonds, although in some cases, nucleic acidanalogs are included that may have at least one different linkage, e.g.,phosphoramidate, phosphorothioate, phosphorodithioate, orO-methylphosphoroamidite linkages (see Eckstein (1992) Oligonucleotidesand Analogues: A Practical Approach Oxford University Press); andpeptide nucleic acid backbones and linkages. For example, peptidenucleic acids (PNA) which includes peptide nucleic acid analogs can beused in the invention.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. As will be appreciated by those in the art, thedepiction of a single strand also defines the sequence of thecomplementary strand; thus the sequences described herein also providethe complement of the sequence. The nucleic acid may be DNA, bothgenomic and cDNA, RNA, or a hybrid, where the nucleic acid may containcombinations of deoxyribo- and ribo-nucleotides, and combinations ofbases, including uracil, adenine, thymine, cytosine, guanine, inosine,xanthine hypoxanthine, isocytosine, isoguanine, etc. “Transcript”typically refers to a naturally occurring RNA, e.g., a pre-mRNA, hnRNA,or mRNA. As used herein, the term “nucleoside” includes nucleotides andnucleoside and nucleotide analogs, and modified nucleosides such asamino modified nucleosides. In addition, “nucleoside” includesnon-naturally occurring analog structures. Thus, e.g., the individualunits of a peptide nucleic acid, each containing a base, are referred toherein as a nucleoside.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into the peptide or used to detect antibodies specificallyreactive with the peptide. The labels may be incorporated into theovarian cancer nucleic acids, proteins and antibodies at any position.Any method known in the art for conjugating the antibody to the labelmay be employed, including those methods described by Hunter, et al.(1962) Nature 144:945-xxx; David, et al. (1974) Biochemistry13:1014-1021; Pain, et al. (1981) J. Immunol. Meth. 40:219-230; andNygren (1982) J. Histochem. and Cytochem. 30:407-412.

An “effector” or “effector moiety” or “effector component” is a moleculethat is bound (or linked, or conjugated), either covalently, through alinker or a chemical bond, or non-covalently, through ionic, van derWaals, electrostatic, or hydrogen bonds, to an antibody. The “effector”can be a variety of molecules including, e.g., detection moietiesincluding radioactive compounds, fluorescent compounds, an enzyme orsubstrate, tags such as epitope tags, a toxin; activatable moieties, achemotherapeutic agent; a lipase; an antibiotic; or a radioisotopeemitting “hard” e.g., beta radiation.

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker or a chemical bond, ornon-covalently, through ionic, van der Waals, electrostatic, or hydrogenbonds to a label such that the presence of the probe may be detected bydetecting the presence of the label bound to the probe. Alternatively,method using high affinity interactions may achieve the same resultswhere one of a pair of binding partners binds to the other, e.g.,biotin, streptavidin.

The term “probe” or a “nucleic acid probe”, as used herein, is definedto be a collection of one or more nucleic acid fragments whosehybridization to a sample can be detected. The probe may be unlabeled orlabeled as described below so that its binding to the target or samplecan be detected. Particularly in the case of arrays, either probe ortarget nucleic acids may be affixed to the array. Whether the arraycomprises “probe” or “target” nucleic acids will be evident from thecontext. Similarly, depending on context, either the probe, the target,or both can be labeled. In some embodiments, the probe may be a memberof an array of spotted nucleic acids. Techniques capable of producinghigh density arrays can also be used for this purpose (see, e.g., Fodor(1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174;Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques23: 120-124; U.S. Pat. No. 5,143,854). One of skill will recognize thatthe precise sequence of the particular probes described herein can bemodified to a certain degree to produce probes that are “substantiallyidentical” to the disclosed probes, but retain the ability tospecifically bind to (i.e., hybridize specifically to) the same targetsor samples as the probe from which they were derived. Such modificationsare specifically covered by reference to the individual probes describedherein.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, e.g., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, e.g., throughthe expression of a recombinant nucleic acid as depicted above.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,e.g., wherein the expression control sequence directs transcription ofthe nucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in “Overview of principles of hybridization and thestrategy of nucleic acid assays” in Tijssen (1993) Hybridization withNucleic Probes (Laboratory Techniques in Biochemistry and MolecularBiology) (vol. 24) Elsevier. Generally, stringent conditions areselected to be about 5-10° C. lower than the thermal melting point (Tm)for the specific sequence at a defined ionic strength pH. The Tm is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions will be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (e.g., 10 to50 nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is typically at least twotimes background, preferably 10 times background hybridization.Exemplary stringent hybridization conditions can be as following: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C is typical for low stringencyamplification, although annealing temperatures may vary between about32-48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90-95° C. for 30-120 sec, an annealing phaselasting 30-120 sec, and an extension phase of about 72° C. for 1-2 min.Protocols and guidelines for low and high stringency amplificationreactions are available, e.g., in Innis, et al. (1990) PCR Protocols: AGuide to Methods and Applications Academic Press, N.Y.

“Inhibitors”, and “modulators” of polynucleotides of the invention areused to refer to molecules or agents that inhibit or modulate theoncogenic effects of the proteins described here. Such agents can beidentified using in vitro and in vivo assays described below. Inhibitorsare compounds that, e.g., bind to, partially or totally block activity,decrease, prevent, delay activation, inactivate, desensitize, or downregulate the activity or expression of the 26#77 proteins describedhere. Such agents include, for example, antisense or inhibitory RNAswhich inhibit expression of the 26#77 gene. Assays for inhibitorsinclude, e.g., expressing the 26#77 protein in vitro, in cells, or cellmembranes, applying test compounds, and then determining the functionaleffects on activity (e.g., changes in growth of the cell). Changes incell growth could be any property associated with a neoplasticphenotype, for example, cell viability, formation of foci, anchorageindependence, semi-solid or soft agar growth, change in contactinhibition or density limitation of growth, loss of growth factor orserum requirements, change in cell morphology, gain or loss ofimmortalization, gain or loss of tumor specific markers, ability to formor suppress tumors when injected into suitable animal hosts, and/orimmortalization of the cell.

“Tumor cell” refers to pre-cancerous, cancerous, and normal cells in atumor.

“Cancer cells,” “transformed” cells or “transformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation is typically associated withphenotypic changes, such as immortalization of cells, aberrant growthcontrol, non-morphological changes, and/or malignancy.

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies) and heteroconjugate antibodies (e.g.,bispecific antibodies). The term “antibody” also includes antigenbinding forms of antibodies, including fragments with antigen-bindingcapability (e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG. See also, PierceCatalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.).See also, e.g., Kuby, J., Immunology, 3^(rd) Ed., W.H. Freeman & Co.,New York (1998). The term also refers to recombinant single chain Fvfragments (scFv). The term antibody also includes-bivalent or bispecificmolecules, diabodies, triabodies, and tetrabodies. Bivalent andbispecific molecules are described in, e.g., Kostelny et al. (1992) JImmunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579,Hollinger et al., 1993, supra, Gruber et al. (1994) J Immunol :5368, Zhuet al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055,Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995)Protein Eng. 8:301.

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, see, e.g., Huse etal., Science 246:1275-1281 (1989); Ward et al, Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

Typically, an immunoglobulin has a heavy and light chain. Each heavy andlight chain contains a constant region and a variable region, (theregions are also known as “domains”). Light and heavy chain variableregions contain four “framework” regions interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs”. The extent of the framework regions and CDRs have beendefined. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

References to “V_(H)” or a “VH” refer to the variable region of animmunoglobulin heavy chain of an antibody, including the heavy chain ofan Fv, scFv, or Fab. References to “V_(L)” or a “VL” refer to thevariable region of an immunoglobulin light chain, including the lightchain of an Fv, scFv, dsFv or Fab.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor proper folding and creation of an active binding site.

A “chimeric antibody” is an immunoglobulin molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

A “humanized antibody” is an immunoglobulin molecule which containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a comparison of Northern and Western blots showingthat both RNA and protein expression of 26#77 are higher in colorectalcancers with an amplified 26#77 gene (T) compared to the patients'normal colorectal tissue (N).

DETAILED DESCRIPTION

This invention provides novel therapeutic and diagnostic methods fortreatment and detection of cancer, as well as methods for screening forcompositions which can be used to treat cancer. As shown below, theinvention is based, at least in part, on the discovery that 26#77 isoverexpressed in colorectal and breast cancer cells. The overexpressionof this gene therefore facilitates progression of carcinogenesis.

Methods of Screening for increased copy number or overexpression of26#77 Genes

In one aspect, 26#77 genes (or their expression levels) are detected indifferent patient samples for which either diagnosis or prognosisinformation is desired. For example, the presence of cancer is evaluatedby a determination of the increased copy number of 26#77 genes in thepatient. Methods of evaluating the presence and/or copy number of aparticular gene or to determine the presence or absence of polymorphismsin the gene are well known to those of skill in the art. For example,hybridization based assays can be used for these purposes.

Hybridization-Based Assays

Hybridization assays can be used to detect copy number of 26#77function. Hybridization-based assays include, but are not limited to,traditional “direct probe” methods such as Southern blots or in situhybridization (e.g., FISH), and “comparative probe” methods such ascomparative genomic hybridization (CGH). The methods can be used in awide variety of formats including, but not limited to substrate—(e.g.membrane or glass) bound methods or array-based approaches as describedbelow.

In a typical in situ hybridization assay, cells or tissue sections arefixed to a solid support, typically a glass slide. If a nucleic acid isto be probed, the cells are typically denatured with heat or alkali. Thecells are then contacted with a hybridization solution at a moderatetemperature to permit annealing of labeled probes specific to thenucleic acid sequence encoding the protein. The targets (e.g., cells)are then typically washed at a predetermined stringency or at anincreasing stringency until an appropriate signal to noise ratio isobtained.

The probes are typically labeled, e.g., with radioisotopes orfluorescent reporters. Preferred probes are sufficiently long so as tospecifically hybridize with the target nucleic acid(s) under stringentconditions. The preferred size range is from about 200 bp to about 1000bases.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-1 DNA is used to block non-specific hybridization.

In comparative genomic hybridization methods a first collection of(sample) nucleic acids (e.g. from a possible tumor) is labeled with afirst label, while a second collection of (control) nucleic acids (e.g.from a healthy cell/tissue) is labeled with a second label. The ratio ofhybridization of the nucleic acids is determined by the ratio of the two(first and second) labels binding to each fiber in the array. Wherethere are chromosomal deletions or multiplications, differences in theratio of the signals from the two labels will be detected and the ratiowill provide a measure of the copy number.

Hybridization protocols suitable for use with the methods of theinvention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234;Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No.430,402; Methods in Molecular Biology, Vol. 33: In Situ HybridizationProtocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In oneparticularly preferred embodiment, the hybridization protocol of Pinkelet al. (1998) Nature Genetics 20: 207-211, or of Kallioniemi (1992)Proc. Natl Acad Sci USA 89:5321-5325 (1992) is used.

A variety of nucleic acid hybridization formats are known to thoseskilled in the art. For example, common formats include sandwich assaysand competition or displacement assays. Hybridization techniques aregenerally described in Hames and Higgins (1985) Nucleic AcidHybridization, A Practical Approach, IRL Press; Gall and Pardue (1969)Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al. (1969) Nature223: 582-587.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system that multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBAO, Cangene, Mississauga,Ontario) and Q Beta Replicase systems.

Typically, labeled signal nucleic acids are used to detecthybridization. The labels may be incorporated by any of a number ofmeans well known to those of skill in the art. Means of attaching labelsto nucleic acids include, for example nick translation, or end-labelingby kinasing of the nucleic acid and subsequent attachment (ligation) ofa linker joining the sample nucleic acid to a label (e.g., afluorophore). A wide variety of linkers for the attachment of labels tonucleic acids are also known. In addition, intercalating dyes andfluorescent nucleotides can also be used.

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include biotin for staining with labeledstreptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescentlabels (e.g., fluorescein, texas red, rhodamine, green fluorescentprotein, and the like, see, e.g., Molecular Probes, Eugene, Oreg., USA),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and calorimetric labels such as colloidal gold (e.g., goldparticles in the 40-80 nm diameter size range scatter green light withhigh efficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

The label may be added to the nucleic acids prior to, or after thehybridization. So called “direct labels” are detectable labels that aredirectly attached to or incorporated into the sample or probe nucleicacids prior to hybridization. In contrast, so called “indirect labels”are joined to the hybrid duplex after hybridization. Often, the indirectlabel is attached to a binding moiety that has been attached to thetarget nucleic acid prior to the hybridization. Thus, for example, thetarget nucleic acid may be biotinylated before the hybridization. Afterhybridization, an avidin-conjugated fluorophore will bind the biotinbearing hybrid duplexes providing a label that is easily detected. For adetailed review of methods of labeling nucleic acids and detectinglabeled hybridized nucleic acids see Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 24: Hybridization With NucleicAcid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

The methods of this invention are particularly well suited toarray-based hybridization formats. For a description of one preferredarray-based hybridization system see Pinkel et al. (1998) NatureGenetics, 20: 207-211.

Arrays are a multiplicity of different “probe” or “target” nucleic acids(or other compounds) attached to one or more surfaces (e.g., solid,membrane, or gel). In a preferred embodiment, the multiplicity ofnucleic acids (or other moieties) is attached to a single contiguoussurface or to a multiplicity of surfaces juxtaposed to each other.

In an array format a large number of different hybridization reactionscan be run essentially “in parallel.” This provides rapid, essentiallysimultaneous, evaluation of a number of hybridizations in a single“experiment”. Methods of performing hybridization reactions in arraybased formats are well known to those of skill in the art (see, e.g.,Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) NatureBiotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958, Pinkelet al. (1998) Nature Genetics 20: 207-211).

Arrays, particularly nucleic acid arrays can be produced according to awide variety of methods well known to those of skill in the art. Forexample, in a simple embodiment, “low density” arrays can simply beproduced by spotting (e.g. by hand using a pipette) different nucleicacids at different locations on a solid support (e.g. a glass surface, amembrane, etc.).

The DNA used to prepare the arrays of the invention is not critical. Forexample the arrays can include genomic DNA, e.g. overlapping clones thatprovide a high resolution scan of a portion of the genome containing thedesired gene, or of the gene itself. Genomic nucleic acids can beobtained from, e.g., HACs, MACs, YACs, BACs, PACs, P1s, cosmids,plasmids, inter-Alu PCR products of genomic clones, restriction digestsof genomic clones, cDNA clones, amplification (e.g., PCR) products, andthe like.

Arrays can also be produced using oligonucleotide synthesis technology.Thus, for example, U.S. Pat. No. 5,143,854 and PCT Patent PublicationNos. WO 90/15070 and 92/10092 teach the use of light-directedcombinatorial synthesis of high density oligonucleotide arrays.

Amplification-Based Assays.

In other embodiments, amplification-based assays can be used to measure26#77 gene copy number in a sample. In such amplification-based assays,the nucleic acid sequences act as a template in an amplificationreaction (e.g. Polymerase Chain Reaction (PCR). In a quantitativeamplification, the amount of amplification product will be proportionalto the amount of template in the original sample. Comparison toappropriate (e.g. healthy tissue) controls provides a measure of thecopy number.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). The known nucleic acidsequence for the genes is sufficient to enable one of skill to routinelyselect primers to amplify any portion of the gene.

Real time PCR is another amplification technique that can be used todetermine gene copy levels or levels of mRNA expression. (See, e.g.,Gibson et al., Genome Research 6:995-1001, 1996; Heid et al., GenomeResearch 6:986-994, 1996). Real-time PCR is a technique that evaluatesthe level of PCR product accumulation during amplification. Thistechnique permits quantitative evaluation of mRNA levels in multiplesamples. For gene copy levels, total genomic DNA is isolated from asample. For mRNA levels, mRNA is extracted from tumor and normal tissueand cDNA is prepared using standard techniques. Real-time PCR can beperformed, for example, using a Perkin Elmer/Applied Biosystems (FosterCity, Calif.) 7700 Prism instrument. Matching primers and fluorescentprobes can be designed for genes of interest using, for example, theprimer express program provided by Perkin Elmer/Applied Biosystems(Foster City, Calif.). Optimal concentrations of primers and probes canbe initially determined by those of ordinary skill in the art, andcontrol (for example, β-actin) primers and probes may be obtainedcommercially from, for example, Perkin Elmer/Applied Biosystems (FosterCity, Calif.). To quantitate the amount of the specific nucleic acid ofinterest in a sample, a standard curve is generated using a control.Standard curves may be generated using the Ct values determined in thereal-time PCR, which are related to the initial concentration of thenucleic acid of interest used in the assay. Standard dilutions rangingfrom 10-10⁶ copies of the gene of interest are generally sufficient. Inaddition, a standard curve is generated for the control sequence. Thispermits standardization of initial content of the nucleic acid ofinterest in a tissue sample to the amount of control for comparisonpurposes.

Other suitable amplification methods include, but are not limited toligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990)Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

Detection of 26#77 Gene Expression

26#77 gene expression level can also be assayed as a marker for cancer.In preferred embodiments, activity of the 26#77 gene is determined by ameasure of gene transcript (e.g. mRNA), by a measure of the quantity oftranslated protein, or by a measure of gene product activity.

Methods of detecting and/or quantifying the gene transcript (mRNA orcDNA) using nucleic acid hybridization techniques are known to those ofskill in the art (see Sambrook et al. supra). For example, one methodfor evaluating the presence, absence, or quantity of mRNA involves aNorthern blot transfer.

The probes can be full length or less than the full length of thenucleic acid sequence encoding the protein. Shorter probes areempirically tested for specificity. Preferably nucleic acid probes are20 bases or longer in length. (See Sambrook et al. for methods ofselecting nucleic acid probe sequences for use in nucleic acidhybridization.) Visualization of the hybridized portions allows thequalitative determination of the presence or absence of mRNA.

In another preferred embodiment, a transcript (e.g., mRNA) can bemeasured using amplification (e.g. PCR) based methods as described abovefor directly assessing copy number of DNA. In a preferred embodiment,transcript level is assessed by using reverse transcription PCR(RT-PCR). In another preferred embodiment, transcript level is assessedby using real-time PCR.

The expression level of an 26#77 gene can also be detected and/orquantified by detecting or quantifying the expressed 26#77 polypeptide.The polypeptide can be detected and quantified by any of a number ofmeans well known to those of skill in the art. These may includeanalytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,or various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting, and the like.Immunohistochemical methods can also be used to detect 26#77 protein.With immunohistochemical staining techniques, a cell sample is prepared,typically by dehydration and fixation, followed by reaction with labeledantibodies specific for the gene product coupled, where the labels areusually visually detectable, such as enzymatic labels, fluorescentlabels, luminescent labels, and the like. A particularly sensitivestaining technique suitable for use in the present invention isdescribed by Hsu et al. (1980) Am. J. Clin. Path. 75:734-738. Theisolated proteins can also be sequenced according to standard techniquesto identify polymorphisms.

The 26#77 polypeptide is detected and/or quantified using any of anumber of well recognized immunological binding assays (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a reviewof the general immunoassays, see also Asai (1993) Methods in CellBiology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NewYork; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

Immunological binding assays (or immunoassays) typically utilize a“capture agent” to specifically bind to and often immobilize the analyte(polypeptide or subsequence). The capture agent is a moiety thatspecifically binds to the analyte. In a preferred embodiment, thecapture agent is an antibody that specifically binds a polypeptide. Theantibody (anti-peptide) may be produced by any of a number of means wellknown to those of skill in the art.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledpolypeptide or a labeled anti-antibody. Alternatively, the labelingagent may be a third moiety, such as another antibody, that specificallybinds to the antibody/polypeptide complex.

In one preferred embodiment, the labeling agent is a second humanantibody bearing a label. Alternatively, the second antibody may lack alabel, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, e.g., asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin. In some embodiments, Western blot analysisis used to detected and or quantify 26#77 protein.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom (1985) J. Immunol., 135: 2589-2542).

26#77 protein can be detected and/or quantified in cells usingimmunocytochemical or immunohistochemical methods. IHC(immunohistochemistry) can be performed on paraffin-embedded tumorblocks using a 26#77-specific antibody. IHC is the method of colormetricor fluorescent detection of archival samples, usually paraffin-embedded,using an antibody that is placed directly on slides cut from theparaffin block. To detect and/or quantify 26#77 in, for example tissueculture cells or cells from a subject that are not embedded in paraffin(for example, hematopoetic cells) ICC (immunocytochemistry) can be used.ICC is like IHC but uses fresh, non-paraffin embedded cells plated ontoslides and then fixed and stained.

Either polyclonal or monoclonal antibodies may be used in theimmunoassays of the invention described herein. Polyclonal antibodiesare preferably raised by multiple injections (e.g. subcutaneous orintramuscular injections) of substantially pure polypeptides orantigenic polypeptides into a suitable non-human mammal. Theantigenicity of peptides can be determined by conventional techniques todetermine the magnitude of the antibody response of an animal that hasbeen immunized with the peptide. Generally, the peptides that are usedto raise the anti-peptide antibodies should generally be those whichinduce production of high titers of antibody with relatively highaffinity for the polypeptide.

Preferably, the antibodies produced will be monoclonal antibodies(“mAb's”). For preparation of monoclonal antibodies, immunization of amouse or rat is preferred. Polyclonal antibodies can also be used.

It is also possible to evaluate an mAb to determine whether it has thesame specificity as a mAb of the invention without undue experimentationby determining whether the mAb being tested prevents a mAb of theinvention from binding to the subject gene product isolated as describedabove. If the mAb being tested competes with the mAb of the invention,as shown by a decrease in binding by the mAb of the invention, then itis likely that the two monoclonal antibodies bind to the same or aclosely related epitope. Still another way to determine whether a mAbhas the specificity of a mAb of the invention is to preincubate the mAbof the invention with an antigen with which it is normally reactive, anddetermine if the mAb being tested is inhibited in its ability to bindthe antigen. If the mAb being tested is inhibited then, in alllikelihood, it has the same, or a closely related, epitopic specificityas the mAb of the invention.

The assays of this invention have immediate utility indetecting/predicting the likelihood of a cancer, in estimating survivalfrom a cancer, in screening for agents that modulate the subject geneproduct activity, and in screening for agents that inhibit cellproliferation.

Methods of Screening for 26#77 Function

Assays for 26#77 function can be designed to detect and/or quantify anyeffect that is indirectly or directly under the influence of the 26#77protein or nucleic acid, e.g., a functional, physical, or chemicaleffect. Such assays can be used to test whether a biological samplecomprises a functional 26#77 protein, to test whether variant 26#77polypeptides retain function, or to identify compounds that modulate26#77 activity in cells.

Typical assays useful in the present invention are those designed totest neoplastic characteristics of cancer cells. These assays includecell growth on soft agar; anchorage dependence; contact inhibition anddensity limitation of growth; cellular proliferation; cell death(apoptosis); cellular transformation; growth factor or serum dependence;tumor specific marker levels; invasiveness into Matrigel; tumor growthand metastasis in vivo; mRNA and protein expression in cells undergoingmetastasis, and other characteristics of cancer cells.

The ability of 26#77 polynucleotides to promote cell growth can also beassessed by introducing the polynucleotides into in cells and assessingthe growth of those cells in vitro or in vivo.

Assays may include those designed to test the ability of test agents tobind the 26#77 protein and thereby modulate its activity. Virtually anyagent can be tested in such an assay. Such agents include, but are notlimited to antibodies, natural or synthetic nucleic acids, natural orsynthetic polypeptides, natural or synthetic lipids, natural orsynthetic small organic molecules, and the like.

Proteins interacting with the peptide or with the protein encoded by thecDNA (e.g., 26#77) can be isolated using a yeast two-hybrid system,mammalian two hybrid system, or phage display screen, etc. Targets soidentified can be further used as bait in these assays to identifyadditional proteins that interact with 26#77 or are downstream of 26#77,which proteins are also targets for drug development (see, e.g., Fieldset al., Nature 340:245 (1989); Vasavada et al., Proc. Nat'l Acad. Sci.USA 88:10686 (1991); Fearon et al., Proc. Nat'l Acad. Sci. USA 89:7958(1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien et al., Proc.Nat'l Acad. Sci. USA 9578 (1991); and U.S. Pat. Nos. 5,283,173,5,667,973, 5,468,614, 5,525,490, and 5,637,463).

Any of the assays for detecting 26#77 binding are amenable to highthroughput screening. High throughput assays for the presence, absence,or quantification of particular nucleic acids or protein products arewell known to those of skill in the art. Similarly, binding assays andreporter gene assays are similarly well known. Thus, for example, U.S.Pat. No. 5,559,410 discloses high throughput screening methods forproteins, U.S. Pat. No. 5,585,639 discloses high throughput screeningmethods for nucleic acid binding (i.e., in arrays), while U.S. Pat. Nos.5,576,220 and 5,541,061 disclose high throughput methods of screeningfor ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols for various high throughput systems.Thus, for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

Recombinant Production of 26#77 Polypeptides

The present invention also provides methods, reagents, and vectorsuseful for expression of 26#77 polypeptides and nucleic acids in vitro.In vitro expression is particularly useful for production of 26#77polypeptides.

Any number of well known host cells can be used for production of 26#77polypeptides. Host cells may be cultured cells, cell lines, cells invivo, and the like. Host cells may be prokaryotic cells such asbacterial cells, (e.g., E. coli), or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells such as CHO, HeLa, and the like.

The particular procedure used to introduce the nucleic acids into a hostcell for expression of the 26#77 protein is not critical to theinvention. Any of the well known procedures for introducing foreignnucleotide sequences into host cells in vitro may be used. These includethe use of calcium phosphate transfection, electroporation,liposome-mediated transfection, injection and microinjection, ballisticmethods, viral particles, virosomes, immunoliposomes, polycation:nucleicacid conjugates, naked DNA, artificial virions, agent-enhanced uptake ofDNA, and the like.

In these embodiments of this invention, 26#77 nucleic acids are insertedinto vectors using standard molecular biological techniques. Vectors maybe used at multiple stages of the practice of the invention, includingfor subcloning nucleic acids encoding components of the 26#77 protein aswell as additional elements controlling protein expression, vectorselectability, etc. Vectors may also be used to maintain or amplify thenucleic acids, for example by inserting the vector into prokaryotic oreukaryotic cells and growing the cells in culture. In addition, vectorsmay be used to introduce and express nucleic acids into cells fortherapeutic or experimental purposes.

A variety of commercially or commonly available vectors and vectornucleic acids can be converted into a vector of the invention by cloninga nucleic acid encoding a 26#77 protein of the invention into thecommercially or commonly available vector. A variety of common vectorssuitable for this purpose are well known in the art.

In a typical embodiment, an 26#77 poynucleotide is placed under thecontrol of a promoter. A nucleic acid is “operably linked” to a promoterwhen it is placed into a functional relationship with the promoter. Forinstance, a promoter or enhancer is operably linked to a coding sequenceif it increases or otherwise regulates the transcription of the codingsequence. Similarly, a “recombinant expression cassette” or simply an“expression cassette” is a nucleic acid construct, generatedrecombinantly or synthetically, with nucleic acid elements that arecapable of effecting expression of a structural gene in hosts compatiblewith such sequences. Expression cassettes include promoters and,optionally, introns, polyadenylation signals, and transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

An extremely wide variety of promoters are well known, and can be usedin the vectors of the invention, depending on the particularapplication. Ordinarily, the promoter selected depends upon the cell inwhich the promoter is to be active. Other expression control sequencessuch as ribosome binding sites, transcription termination sites and thelike are also optionally included. For E. coli, example controlsequences include the T7, trp, or lambda promoters, a ribosome bindingsite and preferably a transcription termination signal. For eukaryoticcells, the control sequences typically include a promoter whichoptionally includes an enhancer derived from immunoglobulin genes, SV40,cytomegalovirus, a retrovirus (e.g., an LTR based promoter) etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences.

For long-term, high-yield production of recombinant proteins, stableexpression will often be desired. For example, cell lines which stablyexpress a 26#77 protein can be prepared using expression vectors of theinvention which contain viral origins of replication or endogenousexpression elements and a selectable marker gene. Following theintroduction of the vector, cells may be allowed to grow for 1-2 days inan enriched media before they are switched to selective media. Thepurpose of the selectable marker is to confer resistance to selection,and its presence allows growth of cells which successfully express theintroduced sequences in selective media. Resistant, stably transfectedcells can be proliferated using tissue culture techniques appropriate tothe cell type. An amplification step, e.g., by administration ofmethyltrexate to cells transfected with a DHFR gene according to methodswell known in the art, can be included.

Kits Use in Diagnostic, Research, and Therapeutic Applications

For use in diagnostic, research, and therapeutic applications disclosedhere, kits are also provided by the invention. In the diagnostic andresearch applications such kits may include any or all of the following:assay reagents, buffers, 26#77-specific nucleic acids or antibodies,hybridization probes and/or primers, and the like. A therapeutic productmay include sterile saline or another pharmaceutically acceptableemulsion and suspension base.

In addition, the kits may include instructional materials containingdirections (i.e., protocols) for the practice of the methods of thisinvention. While the instructional materials typically comprise writtenor printed materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

The present invention also provides for kits for screening formodulators of 26#77. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise one or moreof the following materials: an 26#77 polypeptide or polynucleotide,reaction tubes, and instructions for testing the desired 26#77 function.

A wide variety of kits and components can be prepared according to thepresent invention, depending upon the intended user of the kit and theparticular needs of the user. Diagnosis would typically involveevaluation of a plurality of genes or products. The genes will beselected based on correlations with important parameters in diseasewhich may be identified in historical or outcome data.

Therapeutic Methods

Administration of Inhibitors

As noted above, inhibitors of the invention can be used to treat cancerand other diseases associated with pathological cellular proliferation.The compounds that inhibit 26#77 activity can be administered by avariety of methods including, but not limited to parenteral (e.g.,intravenous, intramuscular, intradermal, intraperitoneal, andsubcutaneous routes), topical, oral, local, or transdermaladministration. These methods can be used for prophylactic and/ortherapeutic treatment. The pharmaceutical compositions can beadministered in a variety of unit dosage forms depending upon the methodof administration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, capsules and lozenges.

The compositions for administration will commonly comprise an inhibitordissolved in a pharmaceutically acceptable carrier, preferably anaqueous carrier. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of active agent in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages are possible in topicaladministration. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington 'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

The compositions containing inhibitors can be administered fortherapeutic or prophylactic treatments. In therapeutic applications,compositions are administered to a patient suffering from a disease(e.g., colon cancer) in an amount sufficient to cure or at leastpartially arrest the disease and its complications. An amount adequateto accomplish this is defined as a “therapeutically effective dose.”Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health. Single ormultiple administrations of the compositions may be administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the agents of this invention to effectively treat thepatient.

Polynucleotide Inhibitors of 26#77 Genes

The activity of 26#77 protein can also be down-regulated, or entirelyinhibited, by the use of antisense polynucleotides, e.g., a nucleic acidcomplementary to, and which can preferably hybridize specifically to, a26#77 encoding mRNA. Binding of the antisense polynucleotide to the mRNAreduces the translation and/or stability of the mRNA.

Antisense polynucleotides can comprise naturally-occurring nucleotides,or synthetic species formed from naturally-occurring subunits or theirclose homologs. Antisense polynucleotides may also have altered sugarmoieties or inter-sugar linkages. Exemplary among these are thephosphorothioate and other sulfur containing species which are known foruse in the art. Analogs are comprehended by this invention so long asthey function effectively to hybridize with the ovarian cancer proteinmRNA. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc.,Natick, Mass.

RNA interference is another mechanism to suppress gene expression in asequence specific manner. See, e.g., Brumelkamp, et al. (2002)Sciencexpress (21Mar., 2002); Sharp (1999) Genes Dev. 13:139-141; andCathew (2001) Curr. Op. Cell Biol. 13:244-248. In mammalian cells,short, e.g., 21 nt, double stranded small interfering RNAs (siRNA) havebeen shown to be effective at inducing an RNAi response. See, e.g.,Elbashir, et al. (2001) Nature 411:494-498.

Ribozymes can also be used to target and inhibit transcription of 26#77nucleotide sequences. A ribozyme is an RNA molecule that catalyticallycleaves other RNA molecules. Different kinds of ribozymes have beendescribed, including group I ribozymes, hammerhead ribozymes, hairpinribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto, et al.(1994) Adv. Pharmacol. 25: 289-317 for a general review of theproperties of different ribozymes).

The polynucleotide inhibitors can be introduced into a cancer cell byany of a number of well known techniques. For example, thepolynucleotide inhibitors can be conjugated to a binding molecule, asdescribed in WO 91/04753. Suitable binding molecules include, but arenot limited to, molecules that bind cell surface receptors on thesurface of the target cancer cell. Preferably, conjugation of thebinding molecule does not substantially interfere with the ability ofthe binding molecule to bind to its corresponding receptor, or blockentry of the inhibitory polynucleotide into the cell. Alternatively, apolynucleotide inhibitor may be introduced into a cell containing thetarget nucleic acid sequence by formation of an polynucleotide-lipidcomplex.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Unknown Gene 26#77 is Amplified and Overexpressed at the RNAand Protein Levels in Primary Human Colorectal Cancers

Chromosome 20q is amplified in approximately 60% of primary humancolorectal cancers. However, no definitive gene target has beenidentified for the amplicon in human colorectal cancers.

Unknown gene 26#77 was originally identified by virtue of its RNAexpression profile in a breast cancer cell line. Recombinant 26#77protein was expressed and used to generate antibodies specific for the26#77protein.

Twelve breast and colorectal cancer cell lines were tested for 26#77 DNAamplification and for RNA and protein levels. The 26#77 gene wasamplified in three of twelve breast and colorectal cancer cell linestested by Southern blot analysis or FISH. Northern blot analysisdemonstrated that 26#77 RNA levels were elevated in nine of the twelvebreast and colorectal cancer cell lines tested.

The 26#77 protein was predominantly localized in the nucleus. Acolorectal cancer cell line (CACO2 cell line) was fractionated intocytoplasmic and nuclear fractions. Western blot analysis with the ant26#77 polyclonal antibody demonstrated that the majority of 26#77protein was found in the nuclear fraction. Immunocytochemical analysisof CACO2 cells also showed that 26#77 was predominantly localized in thenucleus. Similar results were obtained using a breast cancer cell line(BT474) that overexpressed 26#77.

26#77 was also cloned into a tetracycline-inducible vector (fromInvitrogen). The tet-inducible 26#77 vector was then used to transfectNIH 3T3 cells. After induction of 26#77 expression, 26#77 was localizedto the cell nucleus as demonstrated by western blot analysis andimmunocytochemistry.

One hundred and twenty-five primary colorectal cancers with the 20qamplicon were tested for 26#77 gene copy levels. The 26#77 gene wasamplified in 60% of the 125 colorectal cancers tested by Southern blotanalysis or FISH. A subset of the 125 primary colorectal cancers (40samples total) were tested for 26#77 RNA and protein levels. Of the26#77 amplified colorectal cancers in the subset (20 cancers total), allhad elevated levels of 26#77 RNA compared to matched normal colorectaltissue as demonstrated by Northern blot analysis. Exemplary results areshown in FIG. 1. Western blot analysis and immunohistochemistrydemonstrated that 26#77 protein levels were also elevated in thesamples. The results indicate that the 26#77 gene is a target of the 20qamplicon and is an important novel oncogene in human colorectal cancer.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method for determining the presence or absence of a colorectalcancer cell in a patient, the method comprising determining the level ofa target nucleic acid that comprises a sequence at least 99% identicalto SEQ ID NO: 1, wherein the level of the target nucleic acid isdetermined in a colorectal tissue sample from the patient and comparedto the amount of the target nucleic acid in a reference sample, therebydetermining the presence or absence of the colorectal cancer cell in thepatient.
 2. A method for determining the presence or absence of acolorectal cancer cell in a patient, the method comprising determiningthe level of a target nucleic acid that encodes the amino acid sequenceof SEQ ID NO: 2, wherein the level of the target nucleic acid isdetermined in a colorectal tissue sample from the patient and comparedto the amount of the target nucleic acid in a reference sample, therebydetermining the presence or absence of the colorectal cancer cell in thepatient.
 3. The method of claim 1 or claim 2, wherein the colorectaltissue sample comprises isolated nucleic acids.
 4. The method of claim3, further comprising the step of amplifying nucleic acids before thestep of determining the level of the nucleic acid.
 5. The method ofclaim 3, wherein the isolated nucleic acids are mRNA.
 6. The method ofclaim 1 or claim 2, wherein the step of determining the level of targetnucleic acid is carried out using in situ hybridization.
 7. The methodof claim 1 or claim 2, wherein the step of determining the level oftarget nucleic acid is carried out using a labeled nucleic acid probethat selectively hybridizes to SEQ ID NO: 1 under stringenthybridization conditions, wherein the stringent hybridization is carriedout at 65° C. in a buffer comprising 5×SSC and 1% SDS, followed by awash at 65° C. in a buffer comprising 0.2×SSC and 0.1% SDS; or iscarried out at 42° C. in a buffer comprising 50% formamide, 5×SSC, and1%SDS; followed by a wash at 65° C. in a buffer comprising 0.2×SSC and0.1% SDS.
 8. The method of claim 1 or claim 2, wherein the step ofdetermining the level of target nucleic acid is carried out using anucleic acid probe immobilized to a solid support, wherein the probeselectively hybridizes to SEQ ID NO: 1 under stringent hybridizationconditions, wherein the stringent hybridization is carried out at 65° C.in a buffer comprising 5×SSC and 1% SDS, followed by a wash at 65° C. ina buffer comprising 0.2×SSC and 0.1% SDS and 1% SDS; or is carried outat 42° C. in a buffer comprising 50% formamide, 5×SSC, and 1%SDS;followed by a wash at 65° C. in a buffer comprising 0.2×SSC and 0.1%SDS.
 9. The method of claim 1 or claim 2, wherein the step ofdetermining the level of target nucleic acid is carried out usingNorthern blot analysis.
 10. The method of claim 1 or claim 2, whereinthe reference sample is from normal colorectal tissue.
 11. The method ofclaim 1 or claim 2, wherein the patient is undergoing a therapeuticregimen to treat colorectal cancer.
 12. The method of claim 1 or claim2, wherein the patient is suspected of having colorectal cancer.
 13. Amethod for determining the presence or absence of a colorectal cancercell in a patient, the method comprising determining the level of atarget nucleic acid that comprises the sequence of SEQ ID NO: 1, whereinthe level of the target nucleic acid is determined in a colorectaltissue sample from the patient and compared to the amount of the targetnucleic acid in a reference sample, thereby determining the presence orabsence of the colorectal cancer cell in the patient.
 14. The method ofclaim 3, wherein the isolated nucleic acids are DNA.
 15. The method ofclaim 1 or claim 2, wherein the step of determining the level of targetnucleic acid is carried out using a micro array platform.